We are carrying out a program of research aimed at elucidating the molecular mechanisms by which the thymidine analog 5-bromodeoxyuridine (BrdU) affects malignancy, differentiation, viability and DNA-related processes in mammalian cells in general. Through these studies, we hope to identify specific biochemical steps involved in the expression and regulation of malignancy, differentiation and other cellular activities. Most of the studies involve a line of Syrian hamster melanoma cells and mutants derived from it. In studies on the suppression by BrdU of tumorigenicity and pigmentation, we are focusing on the question of whether the effects of BrdU are due to the presence of BrdU in DNA or to effects of BrdU at levels outside DNA. Our results already indicate that BrdU in DNA may not be a primary factor in some cases. However, recent results with cells in which the expression of the transformed phenotype is dependent upon the presence of BrdU suggest that transformation is controlled by the level of BrdU incorporated into DNA. These results raise the possibility of isolating a BrdU-dependent oncogene and elucidating its mechanism of regulation.